CHROMOSOME  NUMBERS  AND 
MORPHOLOGY  IN  TRIFOLIUM 


BY 
HAAKON  WEXELSEN 


University  of  California  Publications  in  Agricultural  Sciences 

Volume  2,  No.  13,  pp.  355-376,  4  figures  in  text 

Issued  May  12,  1928 


University  of  California  Press 
Berkeley,  California 


Cambridge  University  Press 
London,  England 


CHROMOSOME  NUMBERS  AND  MORPHOLOGY 
IN  TRIFOLIUM 


BY 

HAAKON  WEXELSEN* 


CONTENTS 

PAGE 

Introduction 355 

Material    and    methods 356 

Chromosome  numbers  and  morphology 357 

Chromosome  numbers  357 

Variations  in  chromosome  size  in  the  genus 359 

Variations  in  chromosome  size  within  the  species 363 

Chromosome    individuality 365 

Attempts  at   species   crossing *. 370 

Evolution  of  the  chromosome  complexes  in  Trifolium 372 

Summary   375 

Literature  cited  - 376 


INTRODUCTION 

The  genus  Trifolium  is  an  outstanding  genus  within  the  family 
Leguminosae.  It  contains  a  large  number  of  species  which  show 
great  morphological  variation  and  a  wide  geographical  distribution 
and  includes  several  very  important  agricultural  crops,  such  as 
T.  pratense,  T.  repens,  T.  hybrid  urn,  T.  incarnatwn,  and  T.  alexan- 
drinum.  Although  considerable  plant  breeding  work  has  been  carried 
out,  especially  with  T.  pratense,  no  genetic  analysis  of  any  of  these 
species  has  been  made  and  the  cytological  investigations  are  of  very 
recent  date.  The  genetic  analysis  of  other  agricultural  crop  plants 
has  rendered  important  service  to  the  plant  breeder,  and  there  is 
every  reason  to  assume  that  the  same  will  be  the  case  with  the  clovers 
in  which  there  are  a  large  number  of  "good"  genetic  characters. 
It  is  of  importance  that  the  chromosome  situation  in  these  species 
should  be  known  before  genetic  investigations  are  started.  The 
results  of  the  cytological  investigations  have  been  encouraging  to  the 
geneticist  and  plant  breeder  as  they  show  that  in  the  most  important 


*  International  Education  Board  Research  Fellow,  Hjellum,  Norway. 


356  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  2 

agricultural  plants  the  chromosome  numbers  are  fairly  low — 7  and  8 
haploid,  according  to  which  7  and  8  linkage  groups  are  to  be  expected. 
A  genetic  and  cytological  investigation  in  Trifolivm  was  started 
by  the  writer  at  the  Division  of  Genetics  of  the  Department  of  Agri- 
culture, University  of  California,  Berkeley,  in  July,  1926,  and  carried 
on  until  December,  1927.  In  this  paper  will  be  included  only  the 
results  of  the  cytological  investigations  and  the  attempts  at  species 
crossing.  I  take  great  pleasure  in  thanking  Professor  E.  B.  Babcock 
for  laboratory  facilities  and  give  my  best  thanks  to  all  the  members 
of  the  staff  in  the  Division  of  Genetics  for  help  and  advice.  I  am 
greatly  indebted  to  Professor  P.  B.  Kennedy  and  Mrs.  A.  Frederick, 
of  the  Division  of  Agronomy,  for  the  material  and  for  help  in 
identification  of  the  species  used.  Acknowledgment  is  also  given  to 
the  International  Education  Board  for  the  fellowship  granted  to  me. 


MATERIAL  AND  METHODS 

Most  of  the  material  was  grown  from  seeds  furnished  by  Professor 
Kennedy.  The  seeds  of  the  American  species  had  been  obtained  either 
from  plants  growing  wild  or  from  plants  grown  one  generation  in  the 
greenhouse.  Plants  of  these  species  have  been  compared  with  the 
specimens  in  the  Herbarium  of  the  University  of  California,  and  in 
the  collection  of  Professor  Kennedy.  In  the  nomenclature  and  group- 
ing of  these  species  I  have  followed  McDermott  (1910).  The  other 
species  used  were  for  the  most  part  well-known  cultivated  species, 
with  the  exception  of  Trifolium  glomerainm  from  Syzran,  Russia,  and 
T.  subterranum,  which  was  grown  only  to  a  seedling  stage.  These 
seeds  had  been  obtained  from  the  United  States  Department  of 
Agriculture.  The  two  strains  of  T.  repens  used  were  obtained  from 
the  following  sources:  (1)  T.  repens  var.  sylvestre,  wild  white  clover, 
plants  growing  wild  on  the  campus  of  the  University  of  California; 
(2)  T.  repens  var.  giganteum,  Ladino  clover;  Italian  white  clover; 
seeds  from  Vilmorin,  France.  Three  strains  of  T.  pratense,  Italian, 
Late  Swedish,  and  Canadian,  were  obtained  from  the  Central  Experi- 
ment Station,  Ottawa,  Canada. 

The  chromosomes  were  studied  in  somatic  divisions  in  root  tips  ; 
in  two  species  the  reduction  division  in  the  pollen  mother  cells  was 
also  investigated.  For  the  root  tips  the  fixative  of  S.  G.  Nawaschin 
(Karpechenko,  1927,  p.  367)  was  always  used.  Buds  for  the  study 
of  pollen  mother  cells  were  fixed  either  in  Flemming's  medium  or 


1928]        Wexelsen :  Chromosome  Numbers  and  Morphology  in   Trifolium  3o7 

Nawaschin's  fixative.  Most  of  the  plates  were  stained  with  Haiden- 
hain's  iron-haematoxylin,  a  few  with  iodine-gentian-violet  (Huskins, 
1927).  For  Trifolium  the  following  procedure  was  found  to  be  the 
best:  (1)  Root  tips:  70  per  cent  alcohol;  iodine  (5-10  min.)  ;  gentian 
violet  (5-10  min.)  ;  iodine  (30  sec).  (2)  Pollen  mother  cells:  70  per 
cent  alcohol;  gentian-violet  (5-10  min.)  ;  iodine  (30  sec). 

Attempts  were  made  to  study  the  reduction  division  in  pollen 
mother  cells  by  the  aceto-carmine  method,  but  with  no  success.  It  is 
difficult  to  get  the  anthers  out  of  the  small  buds  and  they  are  filled 
with  inclusions  (starch?)  which  apparently  prevent  the  absorption 
of  the  fixative.  The  methods  of  emasculation  and  pollination  will  be 
described  in  the  section  on  ' '  Attempts  at  species  crossing. ' ' 

• 
CHROMOSOME  NUMERS  AND  MORPHOLOGY 

Chromosome  Numbers 

Martin  (1924)  counted  the  chromosomes  in  Trifolium  protense 
and  T.  repens  and  found  the  number  in  both  to  be  12,  haploid. 
Karpechenko  (1925)  examined  the  chromosomes  in  somatic  cells — 
root  tips — of  twenty-four  species  and  found  the  following  series  of 
diploid  chromosome  numbers: 

Diploid   number  of  chromosomes     14       1G       32       48  about  80  about  130 
Number  of  species 8       12         1         1  1  1 

Bleier  (1925)  studied  the  reduction  division  in  eighteen  species  and 
found  the  following  series  of  haploid  chromosome  numbers : 

Haploid  number  of  chromosomes     7  8  9  14  16  48 

Number  of   species 5         8         1(f)     2         —  2 

I  have  obtained  chromosome  numbers  in  ten  native  American  species 
with  the  following  distribution  in  the  groups  given  by  McDermott 
(1910). 

Section  I.    Tridentatae 

2n 

T.  obtusiflorum  Hook.,  2  strains 16 

T.  obtusiflorum  var.  majus  (T.  majus  Greene) 16 

Section  II.     Variegatae 

T.  variegatum  Nutt 16 

T.  wormsl'jolclii  Lehm 48(?)* 

*  But  little  material  was  available  for  fixation  and  the  chromosomes  were  much 
crowded  in  the  cells,  so  that  the  number  could  not  be  obtained  with  certainty. 
There  are  in  figure  lc  47  bodies,  one  of  which  probably  represents  2  chromosomes; 
48  is  very  probably  the  correct  number  of  chromosomes  present. 


358  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  2 

Section  III.     Monantheae 

Section  IV.     Cyathiferae 
T.  microcephalism  Pursh 16 

Section  V.     Vesiculeae 
T.  fucatum  Lindl 16 

T.  fucatum  var.  virescens  (T.  virescens  Greene) 16 

Section  VI.     Bracteolateae 
Section  VII.     Macreae 

2/! 

T.  albopurpureum  T.  and  G 16 

T.  dichotomum  H.  and  A 32 

Section  VIII.    Longifoleae 
T.  reflexion  I». 16 

Section  IX.     Ciliatae 
T.  ciliolatum  Benth.   (T.  ciliatum  Nntt.).  3  6 

The  other  species  counted  are 

In 

T.  pratense  14 

T.  mcarnatv/m   14 

T.  repens,  2  varieties  ...  32 

T.  hybridum  16 

T.  (/lomcratum  16 

T.  minus(^)*  32 

T.  subterranum   16 

T.  alexandrinum    16 

*  The  identification  of  this  .s]hm-h's  is  not  certain,  as  it  was  not  observed  in 
flower.  T.  minus  ought  to  be  studied  anew  to  ascertain  whether  it  has  28 
chromosomes  as  found  by  Bleier  or  32  as  found  by  me.  If  28  is  correct,  this 
represents  the  only  double  species  of  the  7  series. 

The  eighteen  species  counted  by  the  writer  form  t  lie  following 
series  of  diploid  chromosome  numbers : 

Diploid  number  of  chromosomes 14       16       32       48 

Number  of  species  2       12         3         1 

In  Trifolium  ineamatum,  Karpechenko  found  14  somatic  chromo- 
somes and  Bleier  lias  plates  with  both  7  and  8  bivalents  at  heterotypic 
metaphase.  I  found  the  somatic  number  to  be  14,  which  is  probably 
correct  for  this  species.  In  T.  repens,  Karpechenko  found  32  diploid 
chromosomes  and  Bleier  found  14  bivalents  at  first  metaphase.  Erith 
(1924)  counted  the  chromosome  numbers  in  three  varieties  of  T. 
repens.  On  page  113,  Erith  states,  "the  two  cultivated  races  of  white 
clover  have  the  same  number  of  chromosomes   as  the   small   wild 


•2  n 

n 

Karpechenko 

14 

Bleier 

7 

Karpechenko 

14 

Bleier 

7  and  8 

Karpechenko 

32 

Bleier 

14 

Karpechenko 

16 

Bleier 

8 

Bleier 

7 

Bleier 

14 

1928]       Wexelsen:  Chromosome  Numbers  and  Morphology  in  Trifolium  339 

species."  On  page  92  it  is  stated,  "The  diploid  number  of  chromo- 
somes is  sixteen."  Figure  62ft  on  the  same  page  shows,  however, 
a  heterotypic  metaphase  with  16  bodies,  and  figure  62cZ  a  homotypic 
metaphase  with  16  bodies.  From  these  figures  the  conclusion  must 
be  drawn  that  the  forms  investigated  by  Erith  had  32  and  not  16 
as  the  diploid  number.  I  found  the  somatic  number  to  be  32  in  two 
varieties  of  this  species.  In  T.  montarmm,  Bleier  found  9  bivalents 
at  first  metaphase ;  the  count  was  not  certain  and  as  Karpechenko 
found  the  diploid  number  in  montanum  to  be  16,  it  is  probable  that 
there  is  no  species  of  Trifolium  with  18  as  the  diploid  number.  There 
is  now  established  the  following  series  of  haploid  chromosome  numbers 
in  forty-three  species  of  Trifolium  : 

Haploid  number  of  chromosomes....  7  8  14  16  24  about  48  about  130 
Number   of   species 11       23         1         3         2  2  1 

The  basic  numbers  of  this  series  are  7  and  8.  The  7-series  consists 
of  single,  double,  and  possibly  higher  multiple  numbers;  the  8-series 
of  single,  double,  triple,  and  probably  higher  multiples.  This  is  the 
terminology  suggested  by  Belling  (1927)  ;  the  term  single  is  used  for 
the  species  with  the  basic  number,  and  double  and  triple  for  species 
with  two  and  three  times  this  number,  corresponding  to  the  old  terms 
tetraploid  and  hexaploid.  As  for  the  relation  between  chromosome 
numbers  and  the  systematic  classification  of  species,  Karpechenko 
(1925)  states:  "Hence  it  is  evident  that  in  the  process  of  divergence 
of  species  of  clover  certain  chromosome  changes,  undiscerned  by 
observation,  have  greater  significance,  whereas  the  number  of  chromo- 
somes plays  a  subordinate  role."  The  species  studied  by  the  writer 
give  evidence  in  the.  same  direction.  Widely  different  species,  such 
as  Trifolium  variegation  and  T.  reflexum  have  the  same  number  of 
chromosomes,  while  in  one  group  are  found  species  with  16  and  14 
chromosomes.  Among  the  American  species  studied  there  is  no  repre- 
sentative of  the  7-series.  These  species  form  a  regular  multiple  series, 
8-16-24. 

Variations  in  Chromosome  Size  in  the  Genus 

The  chromosomes  in  Trifolium  are  in  general  small.  There  is, 
however,  a  very  large  range  of  variation  in  length  from  about  l^  in 
T.  variegatum  (fig.  Id)  to'  4/t  in  T.  reflexum  (fig.  lb).  There  is  a 
still  greater  difference  in  total  chromosome  volume,  as  illustrated  by 
the  complexes  of  T.  variegatum  (fig.  Id)  and  T.  diehotomum  (fig.  Ik). 


360  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  2 


t^i  %&<?    yp%    v> 


1 


y'A  $k<  A 


Fig.  1.  Somatic  metaphase  figures  from  root  tips  of:  a,  T.  obtusiflorum ;  b,  T. 
ma  jus;  o,  T.  wormskjohtii ;  <1,  T.  variegatum  ;  < ,  T.  microcephalv/mj  f,  T.  fucatum; 
to  the  left  a  plate  with  16  chromosomes,  to  the  right  a  satellited  pair  from 
another  plate;  g,  T.virescens;  h,  T.  albopurpureum;  i,  T.  ciliolatum ;  k,  T.dichoto- 
mum;  I,  T.  reflexum.  All  drawings  for  this  paper  were  made  with  the  aid  of  ;i 
camera  lucida  with  a  Zeiss  18  compensating  ocular  and  a  Leitz  apochromatic 
2  mm.  objective,  N.A.  1.3;  magnification  3650;  figures  not  reduced;  sections 
7/jl,  stained  with  Haidenhain 's  haematoxylin. 


1928J        Wexelsen:  Chromosome  Numbers  and  Morphology  m  Trifolium 


361 


The  species  can  be  grouped  as  follows  according  to  chromosome  size, 
the  species  in  each  group  being  arranged  according  to  increasing  size 
of  the  chromosomes : 


SMALL 

MEDIUM 

1. 

T.  variegatum 

5. 

T.  microcepfialum 

2. 

T.  repens  var.  sylvestre 

6. 

T.  obtusiflorum 

3. 

T.  minus  (?) 

7. 

T.  glomeratum 

4. 

T.  wormsk joldii 

8. 

T  prateme 

9. 

T.  subterranum 

10. 

T.  albopurpureum 

11. 

T.  majus 

12. 

T.  alexandrinum 

13. 

T.  repens  var.  giganteum 

14. 

T.  ciliolatum 

15. 

T.  hybridum 

16. 

T.  virescens 

17. 

T.  fucatum 

LARGE 

18.  T.  incarnatum 

19.  T.  elichotomum 

20.  T.reflexum 


These  groups  are  not  sharply  set  apart ;  if  all  the  chromosomes  in  all 
the  complexes  are  arranged  according  to  size  they  will  form  a  con- 
tinuous series,  but  if  one  looks  at  the  chromosome  complexes  as  such, 
the  complexes  in  the  small  groups  are  distinctly  smaller,  and  those 
in  the  larger  group  larger,  than  the  complexes  in  the  medium  group. 
It  is  not  contended  that  these  groups  have  any  phylogenetic  signifi- 
cance, but  they  may  serve  to  give  a  picture  of  the  situation. 

Bleier  (1925)  discusses  the  question  of  chromosome  size  in  relation 
to  chromosome  number,  nuclear  size,  and  plant  size.  His  discussion 
is  based  on  the  size  of  the  bivalent  chromosomes  at  the  heterotypic 
metaphase  and  on  measurements  of  the  nuclear  diameter  of  the  pollen 
mother  cells  at  the  synaptic  stage.  As  is  pointed  out  by  him  there  is 
great  variation  in  the  size  of  the  metaphase  chromosomes  within  a 
species.  The  same  was  found  in  T.  alexandrinum  in  which  a  large 
number  of  metaphase  plates  were  studied.  In  the  same  way  the 
chromosomes  at  the  somatic  metaphase  show  some  variation  within  the 
species  (see  figs.  2a-  and  2b  of  T.  pratense),  but  the  variation  is  less 
than  in  the  pollen  mother  cells.  Bleier  makes  the  following  statements 
regarding  chromosome  size  in  Trifolium: 

1.  Species  with  the  same  number  of  chromosomes  have  chromosomes  of  different 


2.  The  nuclear  volume  is  not  dependent  upon  the  number  of  chromosomes,  but 
on  the  mass  of  chromatic  substance. 

3.  There  is  no  correlation  between  chromosome  number  and  plant  size,  but 
species  with  a  larger  nuclear  volume  have  larger  growth  than  species  with  small 
volumes. 


362  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  2 


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Fig.  2.  Somatic  metaphase  figures  from  root  tips  of:  a,  T.  pratense;  b,  T. 
pratense;  c,  T.  incarnatum;  d,  T.  alexandrinum ;  e,  T.  hybridum ;  f,  T.  subtcr- 
ranum;  g,  T.  minus;  h,  T.  glomeratum.  Figure  2b  was  stained  with  gentian- 
violet,  the  others  with  Haidenhain's  haematoxylin. 


1928]        Wexelsen:  Chromosome  Numbers  and  Morphology  in  Trifolium  363 

The  first  statement  is  well  illustrated  by  a  comparison  of  the 
chromosomes  in  T.  variegatum  (fig.  Id)  and  T.  reflexum  (fig.  11). 
The  third  statement  may  hold  as  a  general  rule,  but  a  rule  to  which 
there  are  many  exceptions.  T.  obtusiflorum  (fig.  la)  has  16  medium 
to  small  chromosomes,  T.  reflexum,  16  large,  but  the  former  has  the 
largest  plant  size.  That  one  must  be  careful  in  conclusions  based  on 
comparisons  of  chromosome  size  in  species  of  the  same  genus  is  also 
brought  out  by  the  cases  of  intraspecific  variability  in  shape  and  size 
of  chromosomes  discussed  below. 


Variations  in  Chromosome  Size  Within  the  Species 

In  Trifolium  repens,  two  varieties  were  examined  cytologically, 
T.  repens  var.  sylvestre,  wild  white  clover,  and  T.  repens  var.  gigan- 
teum,  Italian  white  clover  or  Lodi  clover,  three  plants  being  studied 
in  each  variety.  The  three  plants  of  giganteum  all  had  chromosomes 
of  about  the  same  size  (fig.  3a,  which  is  a  metaphase  plate  from  a  root 
tip  of  plant  13a ) .  Of  the  three  sylvestre  plants,  plant  la  showed  very 
small  chromosomes,  lb  and  lc  somewhat  larger,  but  all  considerably 
smaller  than  the  chromosomes  of  13a  (fig.  '3b,  c,  d).  Karpechenko 
(1925)  studied  the  somatic  metaphase  in  T.  repens;  he  makes  no  state- 
ment as  to  the  variety  used  but  his  figure  shows  chromosomes  of  the 
same  size  as  those  found  in  giganteum.  Bleier  (1925)  and  Erith  (1924) 
both  studied  pollen  mother  cells  of  repens.  Bleier  says  nothing  about 
which  variety  was  studied,  Erith  states  that  she  counted  giganteum, 
hollandicu.m,  and  sylvestre,  but  does  not  say  anything  about  differ- 
ences in  chromosome  size,  and  it  is  not  clear  from  which  variety  her 
figures  are  taken.  However,  when  the  bivalent  chromosomes  in  her 
plates  (1924,  p.  92,  x  1750)  are  compared  with  those  of  Bleier  (1925, 
p.  618,  x  2150)  it  is  clear  that  the  chromosomes  pictured  by  him  are 
at  least  three  times  as  large  as  those  of  Erith. 

This  case  is  very  interesting  because  giganteum  with  the  large 
chromosomes  is  a  giant  variety,  sylvestre  a  small  variety.  Erith 
(1924)  has  given  detailed  morphological  descriptions  of  the  two 
varieties  which  correspond  to  the  plants  used  by  the  writer.  The 
length  and  breadth  of  the  terminal  leaflet  in  several  plants  of  each 
variety  were  measured  and  the  measurements  for  the  plants  studied 
cytologically  are  given  below.  The  figures  represent  the  average  of 
ten  measurements. 


Leaf 

size  in 

A 

mm. 

Length 

Breadth 

44.1 

32.4 

17.5 

13.8 

12.7 

10.2 

11.4 

10.5 

364  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  2 


Variety  Plant  No. 

giganteum    13a 

sylvestre  la 

lb 
lc 

In  agreement  with  the  results  of  Erith,  I  found  no  difference  in 
flower  size  in  the  two  varieties.  Apparently  the  increase  in  size  in 
giganteum  is  only  in  the  vegetative  parts.  In  accordance  with  this 
is  the  fact  that  the  pollen  is  about  the  same  size  in  the  two  varieties, 
whereas  the  cells  of  the  roots  are  considerably  larger  in  giganteum. 
The  same  was  found  to  hold  true  for  the  stolons  by  Erith  (1924, 
pp.  110-111)  who  states,  "In  older  plants  the  stolons  of  gigcmt&wm 
have  a  diameter  two  to  three  times  that  of  sylvestre,  the  larger 
dimensions  of  the  former  being  due  to  a  greater  number  of  indi- 
vidually larger  cells." 

The  origin  of  giganteum  is  not  known,  but  very  likely  it  arose 
from  sylvestre.  The  genetic  relations  of  the  two  varieties  have  not 
been  determined,  but  some  study  has  been  given  to  chromosome  size 
in  F1  hybrids.  Plant  la  of  sylvestre  was  crossed  with  plant  13a  of 
giganteum  with  la  as  the  mother  plant.  The  Fr  plants  are  still  too 
young  to  make  possible  any  conclusion  as  to  the  behavior  of  plant  size 
in  this  cross.  Two  somatic  metaphases  from  F1  are  pictured  in 
figure  3e  and  /.  The  chromosome  size  is  intermediate,  being  nearer 
to  that  of  the  giganteum  parent.  This  result  suggests  that  the  case 
may  be  one  of  Mendelian  inheritance  of  chromosome  size.  Mendelian 
inheritance  of  a  chromosomal  character  has  been  recorded  by  Lesley 
and  Frost  (1927)  in  Matthiola,  in  which  they  found  that  one 
Mendelian  factor  was  responsible  for  the  difference  in  shape  of  the 
metaphase  chromosomes  of  the  first  meiotic  division.  Because  of  lack 
of  material  of  the  variety  sylvestre,  more  work  must  be  done  to  com- 
plete the  study  of  chromosome  size  in  T.  repens.  As  this  species  is 
self-sterile,  all  the  varieties  are  very  heterozygous,  and  the  plants 
used  by  the  writer  were  very  variable  in  morphological  characters. 
One  might  expect,  therefore,  to  find  many  chromosome  sizes.  It  is 
hoped  that  it  will  be  possible  to  follow  up  this  problem  by  further 
study  of  the  parent  varieties,  the  F1?  and  later  generations.  As  the 
cytological  work  of  the  writer  has  been  discontinued,  at  least  for  some 
time,  it  seems  justifiable  to  give  a  preliminary  account  of  it. 


1928]       Wexelsen:  Chromosome  Numbers  and  Morphology  in  Trifalium  365 


Chromosome  Individuality 

Karpechenko  (1925)  states  that  he  finds  no  chromosome  indi- 
viduality in  the  species  examined  by  him.  In  contrast  to  this  the 
species  reported  upon  here  exhibit  many  differences  in  chromosome 
size  and  shape  within  the  haploid  sets.  The  most  striking  of  these 
is  the  presence  of  satellites  attached  to  the  chromosomes.      In  five 


2fltT 


Fig.  3.  Variations  in  chromosome  size  in  T.  repens.  Somatic  metaphase 
figures  from  root  tips  of:  a,  var.  giganteum,  plant  no.  13a;  b,  var.  sylvestre, 
plant  no.  la;  c  and  d,  of  plants  lb  and  lc  of  the  same  variety;  e,  and  /,  from 
Fj  of  the  cross  la  x  13a.     Stained  with  Haidenhain's  haematoxylin. 

American  species,  representing  three  sections  of  the  genus ;  and  in 
four  European  species,  also  from  three  sections,  there  is  1  pair  of 
satellited  chromosomes.  In  one  species,  Trifoliwm  minus,  there  are 
probably  3  pairs;  in  T.  repens  1  pair  of  satellited  chromosomes  was 
seen  in  one  plate  only  (fig.  3a).  Although  large,  the  satellites  in 
Trifolium  are  often  difficult  to  observe,  because  the  chromosomes  have 
a  tendency  to  stick  together  end  to  end,  and  in  the  same  way  the 
satellites  will  stick  to  the  end  of  the  mother  chromosome.     This  may 


366  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  2 

explain  why  Karpechenko  did  not  find  any  satellites  in  T.  pratense 
and  T.  incarnatum.  in  each  of  which  1  pair  of  conspicuous  satellites 
was  found.  Of  the  species  in  which  no  satellites  were  found,  only 
one,  T.  hybridum,  has  been  investigated  thoroughly  enough  to  state 
with  certainty  that  it  does  not  have  satellites. 

The  existence  of  satellites  was  first  established  by  S.  G.  Nawaschin 
(1912)  in  Galtonia.  Since  that  time  they  have  been  observed  in  many 
species  and  genera.  The  most  outstanding  works  are  those  of  M. 
Nawaschin  (1925,  1926)  on  the  genus  Crepis  and  of  Taylor  (1924, 
1925,  1926)  on  Crepis,  Gasteria,  Allium,  and  other  genera.  In  the 
Leguminosae,  satellites  have  been  found  in  Pisum,  Lathyrus,  and 
Vicia  (Nawaschin,  1925;  Sveshnikova,  1927). 

The  satellites  in  Trifolium  are  large  compared  with  those  observed 
in  most  other  species.  T.  fucatum  (fig.  1/)  seems  to  have  smaller 
satellites,  while  T.  virescens  which  is  nearly  related  to  fucatum,  and 
perhaps  should  be  regarded  as  a  variety  (fig.  lg)  of  this  species,  has 
large  satellites.  This  may  be  a  case  of  the  same  nature  as  that  reported 
by  Nawaschin  (1926)  in  Crepis  dioscoridis,  in  which  he  found  strains 
diffei'ing  in  satellite  size.  It  cannot  be  stated  with  certainty  that 
there  is  a  real  difference  in  satellite  size  in  fucatum  and  virescens. 
There  is  some  variation  in  satellite  size  within  the  strains  and  as  the 
chromosomes  of  fucatum  were  much  crowded  on  the  plates  only  a  few 
observations  of  satellites  were  made  in  this  species.  In  virescens, 
however,  many  observations  were  made,  but  satellites  as  small  as  those 
observed  in  fucatum  were  never  seen. 

A  peculiar  feature  of  these  satellites  is  that  they  sometimes  seem 
to  lie  free  on  the  metaphase  plate  without  any  visible  connection  with 
any  of  the  chromosomes,  as  shown  in  the  metaphase  plate  of  T. 
pratense  (fig.  2a).  The  free  satellites  often  have  a  more  elongated 
shape,  resembling  very  much  a  pair  of  small  chromosomes.  Anyone 
unfamiliar  with  the  material  would  in  such  plates  count  16  chromo- 
somes in  pratense  and  18  in  alcxandrinum.  In  these  two  species  the 
reduction  divisions  in  the  pollen  mother  cells  were  also  studied.  In 
alexandrinum,  many  plates  of  the  first  metaphase  showed  8  bivalents 
(fig.  4a)  and,  in  agreement  with  this,  8  chromosomes  were  found  at 
second  metaphase  (fig.  4fr).  No  trace  of  satellites  was  found  at  these 
stages.  In  T.  pratense,  both  Bleier  and  Karpechenko  found  the 
haploid  number  to  be  7  in  the  reduction  divisions  of  pollen  mother 
cells.  Although  only  a  little  pollen  mother-cell  material  of  T.  pratense 
was   available,    several    good    diakinesis    plates    showed    7    bivalents 


1928]       Wexelsen:  Chromosome  Numbers  and  Morphology  in  Trifolium  367 

(fig.  4c).  As  to  the  nature  of  the  free  satellites  several  interpretations 
can  be  given.  It  is  possible  that  the  fixation  has  failed  to  bring  out 
the  connecting  thread  which  is  really  present;  in  this  case  the 
phenomenon  has  of  course  no  significance.  Against  such  an  interpre- 
tation there  is  the  fact  that  when  the  satellites  appear  to  be  free  they 
ere  usually  found  far  from  any  chromosome,  on  the  outside  of  the 
plate,  while  the  attached  satellites  usually  lie  in  the  middle  of  the 
plate  and  are  connected  with  the  chromosome  by  a  short  and  rather 
thick  thread.  It  may  be,  therefore,  that  the  satellites  sometimes 
become  free  in  the  living  cell ;  in  that  case  they  may  easily  be  lost  in 
the  mitotic  division,  giving  rise  to  "sports"  without  the  satellites. 


*  4' 


Fig.  4.  a,  Heterotypic  division  in  pollen  mother  cell  of  T.  alexandrinwm ; 
b,  homotypic  division  in  same;  e,  diakinesis  in  pollen  mother-cell  of  T.  pratense; 
to  the  right  is  shown  a  bivalent  chromosome  with  attached  satellites  from 
another  diakinesis  plate.  Figure  4»  was  stained  with  Haidenhain's  haema- 
toxylin;  figures  4b  and  c,  with  iodine-gentian- violet. 

This  suggests  the  possibility  that  Karpechenko  worked  with  strains 
of  pratense  and  incarnatum  which  lacked  the  satellites,  This  does 
not  seem  likely  in  the  case  of  pratense,  however,  as  satellites  were 
found  in  four  strains  of  this  species.  Without  making  any  definite 
conclusions  as  to  the  nature  of  the  free  satellites,  it  seems  useful  to 
point  to  the  situation  as  a  possibility  for  geneticists  and  cytologists 
to  bear  in  mind  when  working  with  these  species. 

In  some  of  the  diakinesis  plates  of  pratense  one  bivalent  was  seen 
with  a  pair  of  small  bodies  attached  like  a  pair  of  satellites  (fig.  4c). 
The  observation  was  made  near  the  close  of  the  work  and  no  time  was 
available  to  follow  it  up  by  a  further  study  of  the  reduction  divisions. 
It  seems  very  probable,  however,  that  this  bivalent  corresponds  to  the 
one  pair  of  satellited  chromosomes  to  be  seen  in  figures  of  somatic 
metapha.se.  No  instance  is  known  to  the  writer  in  which  satellites 
have  been  observed  attached  to  bivalent  chromosomes  in  the  reduction 
division.  The  observation  suggests  that  the  maturation  division  in 
species  with  somatic  satellited  chromosomes  should  be  studied,  with 


368  University  of  California  Publications  in  Agricultural  Sciences       [Vol.2 

the  particular  aim  of  tracing  the  satellites  through  the  meiotic  stages. 
If  this  could  be  done  it  would  add  materially  to  the  genetic  signifi- 
cance of  satellites,  as  it  would  show  that  they  are  not  only  a  peculiar 
structure  of  the  somatic  metaphase  chromosomes,  but  are  also  a  part 
of  the  chromosome  which  is  permanently  differentiated  out  from  the 
rest  of  the  chromosome. 

In  many  species  of  plants  it  has  been  found  that  certain  pairs 
of  the  somatic  metaphase  chromosomes  have  definite  and  constant 
constrictions.  In  Trifolium  the  constrictions  are  not  easily  observed 
on  account  of  the  small  size  of  the  chromosomes.  Some  constricted 
pairs  were  established  in  several  species,  but  an  intensive  study  would 
probably  reveal  more  constricted  chromosomes.  The  constrictions  are 
all  subterminal. 

In  general  there  is  not  a  great  variation  in  chromosome  size  within 
the  haploid  sets  in  Trifolium.  Some  species,  however,  show  con- 
spicuous size  differences,  such  as  T.  alexandrinum  (fig.  2d),  T.  incar- 
natum  (fig.  2c),  T.  hybridum  (fig.  2e),  and  T.  reflexum  (fig.  1/). 
The  chromosome  morphology  of  the  species  was  studied  with  two  main 
objectives  in  view: 

1.  In  order  to  be  able  to  distinguish  each  member  or  at  least  the 
groups  of  a  haploid  set.  This  is  usually  done  in  combination  with  a 
genetic  analysis,  by  which  method  it  is  possible  to  assign  a  particular 
gene  to  a  particular  chromosome. 

2.  In  order  to  compare  the  chromosome  complexes  in  species  of 
the  same  genus  and  by  this  method  to  study  their  relationship  and 
origin.  It  has  now  come  to  be  used  also  in  practical  plant  taxonomy 
to  determine,  in  cases  of  doubt,  whether  nearly  related  forms  should 
be  ranked  as  distinct  species.  The  first  problem  has  been  the  chief 
concern  of  the  present  study  of  chromosome  morphology  in  species 
which  seemed  the  most  promising  from  a  genetic  standpoint.  In  these 
species  the  following  features  of  chromosome  individuality  have  been 
revealed : 

T.  pratensc: 

1  pair  of  satellited  chromosomes,  G  pairs  of  about  equal  size,  without 
visible  constrictions. 
T.  inearnatum  : 

1  pair  of  satellited  chromosomes. 

1  pair  of  large,  constricted  chromosomes. 

2  pairs  of  medium,  constricted  chromosomes. 

3  pairs  of  medium  chromosomes  without  visible  constrictions. 
1  pair  of  small,  constricted  chromosomes. 


1928]       Wexelscn:  Chromosomt  Numbers  and  Morphology  in  Tri folium  3C9 

T.  alexandrinum : 

1  pair  of  satellited  chromosomes. 

1  pair  of  large,  constricted  chromosomes. 

3  pairs  of  medium,  constricted  chromosomes. 

2  pairs  of  medium  chromosomes  without  visible  constrictions. 
1  pair  of  small,  constricted  chromosomes. 

The  complexes  in  the  last  two  species  are  similar  but  alexandrinum  has  one 
more  pair  of  medium  sized  chromosomes. 
T.  hybridum: 

6  pairs  of  large  chromosomes,  at  least  three  pairs  with  constrictions. 

1  pair  of  very  small  chromosomes. 

1  pair  of  small,  constricted  chromosomes. 

The  smallest  pair  of  chromosomes  in  hybridum  is  of  the  same  size 
as  the  satellites  of  pratense,  and  the  plates  of  hybridum  resemble  very 
much  the  plates  of  pratense  in  which  the  satellites  have  no  visible 
connection  with  the  chromosomes. 

In  T.  repens,  Bleier  found  in  the  first  metaphase  of  the  reduc- 
tion division  4  small  and  10  large  bivalents.  The  somatic  plates 
studied  also  indicate  that  there  is  one  group  of  small  and  one  of  large 
chromosomes,  but  it  is  very  difficult  to  get  32  chromosomes,  all  lying 
flat  on  the  plate,  so  that  nothing  can  be  said  with  certainty  as  to  the 
number  of  chromosomes  in  each  group.  It  may  be  of  interest  to  note 
that  in  the  two  nearly  related  species,  hybridum  and  repens,  we  find 
in  the  former  2  pairs  of  small  chromosomes  and  in  the  latter  prob- 
ably 4  pairs.  In  one  plate  of  repens  (fig.  3c)  1  pair  of  satellited 
chromosomes  was  seen,  so  it  is  probable  that  repens  has  satellited 
chromosomes.  As  hybridum  has  no  satellites  this  would  mean  that 
repens  has  not  simply  twice  the  complex  of  hybridium. 

Outstanding  in  their  chromosome  morphology  are  also  T.  minus 
and  T.  reflexum.  It  is  interesting  that  the  double  species,  T.  minus 
(32  diploid),  has  probably  3  pairs  of  satellited  chromosomes  (fig.  2g) 
while  no  single  species  has  been  found  with  more  than  one  pair  of 
satellites. 

T.  reflexum  (fig.  1/)  exhibits  a  chromosome  complex  different  from 
all  other  investigated  species,  with  5  pairs  of  large  constricted 
chromosomes  and  3  pairs  of  smaller  chromosomes. 


370  University  of  California  Publications  in  Agricultural  Scie7ices       [Vol.  2 


ATTEMPTS  AT  SPECIES  CROSSING 

The  cases  of  recorded  species  hybrids  in  Trifolium  are  listed  by 
Karpechenko  (1925)  and  Bleier  (1925).  In  all  cases  one  of  the 
parents  was  a  species  with  a  high  chromosome  number,  T.  paunomcum 
(130)  or  T.  medium  (80).  Hitherto,  however,  no  report  has  been 
given  of  an  Fx  hybrid  which  has  been  cytologically  investigated. 
Crosses  were  attempted  between  nine  species  in  eighteen  different 
combinations.  The  material  was  the  same  as  that  used  for  cytological 
investigations.     The  methods  used  were  mainly  two : 

1.  The  heads  were  enclosed  in  paper  bags  before  any  flower  had 
opened.  Emasculation  was  performed  when  the  head  was  about  half 
developed.  The  flowers  which  were  either  too  old  or  two  young  were 
cut  away  and  in  the  rest  of  the  flowers  the  anthers  were  removed  with 
a  forceps.  This  operation  is  fairly  easy  in  the  species  with  large 
flowers,  but  difficult  in  the  small-flowered  ones.  The  flowers  were 
mostly  pollinated  immediately  after  emasculation,  in  some  cases  the 
next  day.  All  the  instruments  used  were  washed  in  alcohol  frequently 
during  the  work  and  only  very  few  cases  of  selfing  occurred. 

2.  Using  plants  of  self-sterile  species  as  mother  plants,  the  pollen 
from  other  species  was  applied  without  emasculation  to  the  stigma  in 
flowers  which  had  been  bagged  before  opening. 

Cross  Number  of  flowers  crossed 

1.  T.  pratense  x  T.  incarnatum  950 

2.  T.  pratense  x  T.  repens  327 

3.  T.  pratense  x  T.  hybridum  284 

4.  T.  pratense  x  T.  fucatum  61 

5.  T.  pratense  x  T.  virescens  ...  154 

6.  T.  pratense  x  T.  obtus-iflorum  32 

7.  T.  repens  x  T.  hybridum  129 

8.  T.  repens  x  T.  incarnatum  20 

9.  T.  incarnatum  x  T.  alexandrinum  186 

10.  T.  incarnatum  x  T.  reflexum  26 

11.  T.  incarnatum  x  T.  obtusiflorum 290 

12.  T.  incarnatum  x  T.  virescens  92 

13.  T.  virescens  x  T.  fucatum  33 

14.  T.  virescens  x  T.  obtusiflorum  6 

15.  T.  vvrcseens  x  T.  reflexum  — 

16.  T.  obtusiflorum  x  T.  fucatum  6 

17.  T.  obtus-iflorum  x  T.  reflexum  15 

18.  T.  reflexum  x  T.  ciliolatum  11 


1928J       Wexelsen:  Chromosome  Numbers-  and  Morphology  in  Trifolium  371 

With  both  methods  the  results  were  completely  negative ;  a  few- 
seeds  obtained  by  either  method  proved  to  be  due  to  selfing.  Outside 
of  these  there  seemed  to  be  no  seed  development  at  all.  Below  are 
given  the  combinations  which  were  tried  and  the  number  of  flowers 
crossed  in  each  combination.  All  the  crosses  were  made  reciprocally 
except  in  4,  8,  10,  14,  15,  and  18. 

In  crosses  1,  2,  3,  5,  7,  9,  and  11  the  number  of  trials  is  large 
enough  to  allow  the  statement  that  hybrids  between  these  species  are 
not  easily  obtained. 

In  pratense,  repens,  hybridum,  and  virescens  intraspecific  crosses 
were  made  and  seeds  easily  obtained,  so  the  negative  results  are  not 
due  to  faulty  technique.  T.  fucatum  and  T.  virescens  are  two  very 
nearly  related  species  or  varieties  of  the  same  species  which  did  not 
seem  to  cross.  The  number  of  flowers  crossed  is  not  large,  but  when 
crossing  plants  within  virescens  seeds  were  easily  obtained.  These 
results  do  not,  of  course,  allow  the  conclusion  that  hybrids  cannot  be 
obtained  between  these  species,  but  they  suggest,  in  agreement  with 
the  residts  of  other  investigators,  that  interspecific  hybrids  are  difficult 
to  secure. 

In  case  of  the  crosses  T.  pratense  x  T.  repens,  and  T.  hybridum, 
respectively,  it  was  attempted,  using  the  method  described  by  Martin 
(1913),  to  study  the  behavior  of  the  pollen  of  repens  and  hybridum 
on  the  stigma  of  pratense.  Flowers  of  pratense  were  emasculated, 
pollinated  immediately,  and  the  stigmas  picked  out  for  observation 
after  18,  24,  48,  and  72  hours.  The  stigmas  were  mounted  on  a  slide 
in  aceto-carmine  and  a  slight  pressure  was  exerted  on  the  coverglass 
to  flatten  the  stigma,  The  pollen  both  of  repens  and  hybridum 
germinated  readily  on  the  stigma  of  pratense,  but  it  was  not  found 
possible  to  follow  the  pollen  tube  growth  through  the  style  by  Martin 's 
method.  Nothing,  therefore,  was  ascertained  as  to  what  happened  to 
the  pollen  tubes.  It  may  be  that  the  situation  is  the  same  as  in  self- 
sterile  species  of  Trifolium  in  which,  when  selfed,  the  pollen  will 
germinate,  but  the  pollen  tube  growth  is  too  slow  to  reach  the  ovary. 


372  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  2 


EVOLUTION   OF   THE    CHROMOSOME    COMPLEXES 
IN  TRIFOLIUM 

The  chromosome  complexes  in  many  genera  are  now  studied  with 
the  aim  of  tracing  the  relationship  between  the  species  and  of  finding 
the  way  in  which  the  evolution  of  the  species  has  proceeded.  Attempts 
are  also  made  to  base  the  classification  of  species  on  chromosome 
morphology.  For  Vicia  Sveshnikova  (1927)  has  worked  out  a  key 
based  on  chromosome  morphology  and  finds  that  it  corresponds 
very  nearly  to  the  key  worked  out  by  Ascherson  based  on  external 
morphology.  In  Trifolium  it  is  evident  that  there  is  no  such  paral- 
lelism in  the  differentiation  of  the  chromosome  complexes  and  of  the 
external  morphology  of  the  species.  Species  which  are  far  removed 
taxonomically  and  very  different  in  their  morphology  have  very 
similar  chromosome  complexes;  for  instance,  the  European  species, 
T.  glomeratum,  and  the  California^  species,  T.  obtusiflorum.  On  the 
other  hand,  we  find  nearly  related  species  with  very  different  chromo- 
some complexes.  The  wild  red  clover,  T.  pratense,  is  very  similar  to 
T.  medium,  but  the  former  has  14  and  the  latter  about  130  chromo- 
somes. Furthermore,  though  the  number  is  the  same,  the  shape  and 
the  size  of  the  chromosomes  may  be  different.  T.  pratense  and 
T.  incarnatum  are  placed  in  the  same  subsection  of  the  section, 
Eulagopus,  but  the  chromosome  complexes  are  very  unlike.  In  T. 
alexandrinum  and  T.  incarnatum  we  have  two  species  differing  in 
external  morphology  and  in  chromosome  number  (16  and  14)  but 
very  similar  as  regards  the  shape  and  size  of  the  chromosomes, 
alexandrinum  having  an  extra  pair  of  medium  sized  chromosomes. 
The  situation  in  Trifolium  suggests  that  it  will  not  be  easy  on  the 
basis  of  chromosome  morphology  to  trace  the  mutual  relationship  and 
origin  of  the  species  in  this  genus.  The  basis  for  such  a  study  must 
be  the  possibility  of  establishing  certain  types  of  chromosomes,  which 
can  be  identified  in  related  species.  Nawaschin's  (1925)  work  on  the 
genus  Crepis  is  of  this  type.  In  ten  species  with  3,  4,  and  5  pairs 
of  chromosomes  he  established  five  types  of  chromosomes,  one  of  which 
was  a  satellited  chromosome.  In  the  summary  Nawaschin  states, 
"Es  wurde  von  mir  festgestellt  dass  dieselbe  homologischen  Typen 
und  Formen  der  Chromosomen  in  den  Chromosomsatzen  aller  unter- 
suchten  Arten  hervortreten. "    In  Trifolium  ten  species  at  least  have 


1£)28]        Wexelsen:  Chromosome  Numbers  and  Morphology  in  Trifolium  373 

1  pair  of  satellited  chromosomes;  but  considering  the  fact  that  species 
from  very  different  sections  have  the  satellites  and  that,  on  the  other 
hand,  species  with  and  without  satellites  occur  in  the  same  section, 
this  feature  does  not  help  much  in  establishing  any  relationship 
between  the  species.  It  does  not  seem  safe,  either,  to  take  chromosome 
size  in  general  as  an  evidence  of  relationship,  when  we  remember 
that  the  one  species,  T.  repens,  includes  in  itself  almost  the  total 
variability  in  chromosome  size  in  the  genus.  It  is  apparently  only  in 
the  narrowest  taxonomic  groups  that  there  is  a  similarity  in  chromo- 
some morphology  which  points  to  common  descent,  and  it  is  probably 
here  that  the  study  of  the  chromosomes  may  be  of  help  to  the 
taxonomist.  Some  facts  pointing  to  this  conclusion  may  be  mentioned. 
T.  variegatum  in  the  section  Variegatae  has  16  very  small  chromo- 
somes. In  the  same  section  is  T.  wormskjoldii  with  48  equally  small 
chromosomes.  This  suggests  that  these  two  species,  in  regard  to  their 
chromosomes,  are  more  nearly  related  than  the  Californian  clovers  of 
other  sections.  The  two  nearly  related  forms,  T.  fucatum  and  T. 
virescens,  have  almost  identical  chromosome  complexes.  The  chromo- 
some sizes  of  the  two  related  species,  T.  hybridum  (16  diploid)  and 
T.  repens  (32  diploid),  indicate  that  the  latter  may  have  a  complex 
which  is  twice  that  of  the  former. 

The  situation  in  Trifolium  is  interesting  because  it  seems  to  demon- 
strate another  type  of  differentiation  of  the  chromosome  complexes 
than  is  found  in  many  other  genera-studied.  The  genera  which  have 
been  most  intensively  investigated  cytologically  are  those  in  which 
interspecific  hybridization  has  been  carried  out.  There  has  been,  then, 
a  preference  for  genera  in  which  interspecific  hybrids  are  fairly  easily 
obtained,  and  in  which  such  hybrids  are  common  in  nature.  This  has 
led  some  investigators  to  emphasize  hybridization  as  the  only  factor 
in  species  differentiation,  and  it  may  perhaps  not  be  out  of  the  way 
to  hold  forth  that  there  may  be  other  ways  of  evolution  of  species. 
It  seems  only  fair  to  do  so  in  connection  with  this  study  in  Trifolium, 
because  all  evidence  suggests  that  hybridization  has  not  played  a 
dominant  role  in  the  differentiation  of  this  genus.  Hybrids  are  very 
rare  in  nature,  if,  indeed,  ever  observed,  and  no  hybrids  have  been 
obtained  in  experiments.  Taking  into  account  only  the  external 
morphology  of  the  chromosomes,  in  Trifolium  no  certain  instance  of 
"homologous"  chromosomes  in  different  species  is  known,  whether 
in  the  form  of  one  single  chromosome,  a  group  of  chromosomes,  or  a 
complete  haploid  set.     The  existence  of  a  satellited  chromosome  pair 


374  University  of  California  Publications  in  Agricultural  Sciences       [Vol.  2 

in  many  species  cannot,  in  the  opinion  of  the  writer,  be  taken  as 
evidence  in  this  direction.  The  fact  that  the  satellited  chromosome 
pair  varies  in  size  in  the  different  species  according'  to  the  general 
size  of  the  chromosome  complex  should  make  one  cautious  in  drawing 
any  such  conclusions  and  this  is  still  clearer  when  the  distribution 
of  the  satellited  chromosomes  is  taken  into  account.  In  the  section 
Euamoria  we  find  T.  hybridum  without  satellites,  and  T.  gJomeratum 
with  satellites  which  resemble  the  satellites  in  T.  obtusiflarum  from 
a  very  different  section.  We  are  not  at  all  justified  in  concluding 
that  glomeratum  and  obtusiflorum  have  obtained  their  chromosome 
complex  from  a  common  source.  In  genera  in  which  interspecific 
hybridization  is  common  there  have  been  found  not  only  polyploid 
series  of  chromosome  numbers,  but  all  intermediate  numbers  as  well. 
A  typical  genus  of  this  kind  is  Viola  (Clausen,  1927),  which  in  the 
section  Melanium  has  the  following  haploid  numbers  of  chromosomes: 
7,  10,  11,  12,  13,  17,  18,  20,  24,  30. 

In  TrifoJium  simple  polyploid  series  without  intermediate  numbers 
are  found ;  2n=  16,  32,  48  ;  and  14,  28,  ( ?). 

It  may  then  perhaps  be  justifiable  to  give  a  suggestion  as  to  the 
evolution  of  the  chromosome  complexes  in  TrifoJium.  This  genus  pre- 
sents a  very  clear  demonstration  of  parallel  variation,  i.e.,  we  find  in 
species  belonging  to  very  different  sections  that  evolution  has  pro- 
ceeded along  parallel  lines.  It  seems  better  in  accordance  with  the 
facts  to  ascribe  this  parallel  variation  to  parallel  independent  muta- 
tions than  to  a  common  descent.  This  is  supported  by  the  fact  that 
we  find  in  many  species  similar  variations  from  the  wild  type.  In 
species  from  different  sections  is  found  the  mutant  form  characterized 
by  the  absence  of  leaf  spots.  In  T .  prattusc  is  found  a  variation  from 
the  normal  red  flower  color  to  white ;  in  T.  repens  a  variation  from 
white  to  red,  but  the  red  and  white  flower  color  in  pratense  and  in 
repens  is  a  genetically  different  character.  The  chromosome  complexes 
show  the  same  picture  as  the  morphological  characters.  The  presence 
of  one  pair  of  satellited  chromosomes  should  be  due,  then,  to 
independent  parallel  mutations  and  not  to  the  fact  that  they  have 
been  derived  from  a  common  source.  This  suggestion  as  to  the  way 
in  which  the  chromosome  complexes  in  TrifoJium  have  been  differ- 
entiated is  supported  also  by  the  variation  in  chromosome  size 
described  in  T.  repens,  which  is  just  an  example  of  that  kind  of 
variation  which  the  hypothesis  supposes  to  take  place.  The  great 
variability  in  chromosome  morphology  in  TrifoJium  is  held  to  be  due, 


1928]        Wexelstn:  Chromosome  Numbers  and  Morphology  in   Trifolium  375 

then,  to  mutational  changes  in  species  isolated  by  interspecific  sterility. 
It  is  not  contended  that  crosses  have  not  taken  place  in  this  genus, 
but  it  is  held  that  the  species  have  been  thus  isolated  for  a  long  time 
and  that  many  mutations  have  occurred. 


SUMMARY 

1.  Somatic  chromosome  numbers  have  been  obtained  in  eighteen 
species  of  Trifolium ,  twelve  of  which  had  not  been  counted  before ; 
the  reduction  division  was  studied  in  two  species. 

2.  The  ten  American  species  studied  have  the  diploid  numbers 
16,  32,  48.  No  representative  is  found  of  the  7 -series  which  is  found 
in  European  species  of  Trifolium. 

3.  The  chromosomes  of  Trifolium  are  in  general  small,  but  they 
exhibit  a  great  variation  in  size,  both  as  to  single  chromosomes  and 
to  total  amount  of  chromatin. 

4.  In  T.  repens  L.  the  two  varieties  giganteum  and  sylvestre  proved 
to  have  chromosomes  of  different  size.  Giganteum  is  a  giant  variety 
and  has  large  chromosomes ;  sylvestre  is  a  small  variety  and  has  small 
chromosomes.  Fx  plants  between  these  two  varieties  showed  chromo- 
somes of  intermediate  size. 

5.  Ten  species  from  several  sections  of  the  genus  have  been  shown 
to  have  1  pair  of  satellited  chromosomes  and  one  species  probably 
has  3  such  pairs. 

6.  The  satellites  are  in  some  plates  without  visible  connection  with 
any  chromosome  and  appear  like  an  extra  pair  of  small  chromosomes. 
In  a  few  diakinesis  plates  of  T.  pratense  were  observed  bodies  which 
must  be  interpreted  as  1  pair  of  satellites  attached  to  a  bivalent 
chromosome. 

7.  On  the  basis  of  satellites,  constrictions,  and  chromosome  size, 
a  scheme  of  chromosome  morphology  has  been  given  for  some  of  the 
species. 

8.  Species  crosses  were  attempted  between  nine  species  in  eighteen 
different  combinations,  but  with  completely  negative  results. 

9.  The  suggestion  is  made  that  the  diversity  of  chromosome  com- 
plexes in  Trifolium  is  a  result  of  mutational  changes  in  species  which 
have  become  isolated  by  intersterility  rather  than  the  result  of 
hybridization. 


376 


University  of  California  Publications  in  Agricultural  Sciences       [Vol. 


LITERATURE  CITED 

1927.     The  nomenclature  of  chromosome  groups.     Nature,  vol.  119   p    9*6 
Bleier,  H.  "' 

1925.     Chromosomeustudien  bei  der  Gattung  Trifolium.    Jahrb   f    wiss   Bot 
vol.  64,  pp.  604-36. 
Clausen,  J. 

1927.     Chromosome   number   and    the    relationship   of   species   in    the   genus 
Viola.     Annals  of  Bot.,  vol.  41,  pp.  677-714 
Lrith,  A.  G. 

1924.  White  clover  (T.  repens  L.).     London,  Duckworth  and  Co.     150  pp 
Huskins,  C.  L.  * i  - 

1927.     On  the  genetics  and   cytology  of  fatuoid   or  false   wild   oats.     Jour 
Genetics,  vol.  15,  pp.  315-64. 
Karpechenko,  G.  D. 

1925.  Karyologisehe   Studien    fiber   die   Gattung   Trifolium.     Bull.   Applied 

Bot.  and  Plant  Breeding,  vol.  14,  pp.  1-9. 
1927.     The  production  of  polyploid  gametes  in  hvbrids.     Hereditas    vol    '» 
pp.  349-68.  '         '  *' 

Lesley,  M.'  M.,  and  Frost,  H.  B. 

1927.     Mendelian  inheritance  of  chromosome  shape  in  MaUKiola.     Genetics 
vol.  12,  pp.  449-60.  ' 

Martin,  J.  N. 

1913.  The   physiology    of   the    pollen    of    T.   pratcnse.      Bot.    Gaz      vol     56 

pp.  112-26.  '  ' 

1914.  Comparative  morphology  of  some  Leguminosae.     Bot    Gaz     vol    58 

pp.  154-66.  '  ''  ' 

McDermott,  L.  F. 

1910.     An  illustrated  key  to  the  North  American  species  of  Trifolium.     San 
Francisco,  Cunningham,  Curtis  and  Welch      325  pp 
Nawaschibt,  M. 

1925.  Morphologische  Kernstudien  der  Crepis-Arten  in  Bezug  auf  die  Art 

bildung.     Zeitschr.  f.  Zellforschung  und  Mikroskopische  Anatomie 
vol.  2,  pp.  98-111. 

1926.  Variability    des   Zellkerns   bei   Crepis-Arten    in    Bezug   auf   die   Art- 

bildung.     Ibid.,  vol.  4,  pp.  171-215. 
Xawaschin,  S.  G. 

1912.  rber  den  Dimorpbismus  der  Kerne  in  den  somatischen  Zellen  bei 
GaZtonw  candicans.  Bull.  Imp.  Acad.  Sei.  St.  Petersboum  ser  6 
vol.  6,  pp.  373-85.  s'  ' 

Sveshnikova,  J.  X. 

1927.  Karyological  studies  in   Vicia.     Bull.  Applied  Bot.  and   Plant  Breed- 

ing, vol.  17,  pp.  37-72. 
Taylor,  W.  P. 

1924.  Cytologiea]  studies  on  Gasteria.  I.  Chromosome  shape  and  individu- 
ality.    Am.  Jour.  Bot.,  vol.  11,  pp.  51-59. 

1025.  Chromosome  constrictions  as  distinguishing  characteristics  in  plants 
Ibid.,  vol.  12,  pp.  238-44. 

1926.  Chromosome  morphology  in  Fritillaria,  Ahtroemeria.  Silphium  and 
other  genera.     Ibid.,  vol.  13,  pp.  180-93. 


